跳轉至內容
Merck
  • Studying physical chromatin interactions in plants using Chromosome Conformation Capture (3C).

Studying physical chromatin interactions in plants using Chromosome Conformation Capture (3C).

Nature protocols (2009-08-01)
Marieke Louwers, Erik Splinter, Roel van Driel, Wouter de Laat, Maike Stam
摘要

Gene regulation in higher eukaryotes frequently involves physical interactions between genomic sequence elements tens of kilobases apart on the same chromosome but can also entail interactions between different chromosomes. Chromosome Conformation Capture (3C) is a powerful tool to identify such interactions. 3C technology is based on formaldehyde crosslinking of chromatin, followed by restriction digestion and intramolecular ligation. Quantitative detection of ligation products by PCR (qPCR; not discussed in this protocol) provides insight into the interaction frequencies between chromosomal fragments and thereby the spatial organization of a genomic region. Detailed 3C protocols have been published for yeast and mammals. However, these protocols cannot simply be transferred to plant tissues. In this paper, we provide a maize-specific 3C protocol and present a general strategy to systematically optimize the protocol for other plants. Once the technique and appropriate controls are established, the 3C procedure (including qPCR) can be completed in 5-7 d.

材料
產品編號
品牌
產品描述

Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
DL-二硫苏糖醇, for molecular biology, ≥98% (HPLC), ≥99% (titration)
Sigma-Aldrich
苯甲磺酰氟, ≥98.5% (GC)
Roche
RNase A, from bovine pancreas
Sigma-Aldrich
苯酚 溶液, Equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA, BioReagent, for molecular biology
Roche
DAPI, 4′,6-Diamidine-2′-phenylindole dihydrochloride
Sigma-Aldrich
乙酸钠, anhydrous, for molecular biology, ≥99%